Cell expansion system and methods of use

ABSTRACT

Cell expansion systems and methods of use are provided. The cell expansion systems generally include a hollow fiber cell growth chamber, and first and second circulation loops (intracapillary loops and extracapillary loops) associated with the interior of the hollow fibers and exterior of the hollow fibers, respectively. Detachable flow circuits and methods of expanding cells are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 12/042,798, entitled, “CELL EXPANSION SYSTEM ANDMETHODS OF USE,” filed Mar. 5, 2008 and issued as U.S. Pat. No.8,309,347 on Nov. 13, 2012, which claims benefit under 35 U.S.C. §119(e)to U.S. Provisional Application Nos. 60/892,908, filed Mar. 5, 2007;60/892,966, filed Mar. 5, 2007; 60/892,911, filed Mar. 5, 2007;60/892,977, filed Mar. 5, 2007; 60/911,393, filed Apr. 12, 2007;60/911,594, filed Apr. 13, 2007; and 60/971,494, filed Sep. 11, 2007.Each of these applications is incorporated herein by reference in itsentirety.

FIELD

The present disclosure relates to cell expansion systems (CESs),associated cell growth chambers, and methods of using the same.

BACKGROUND

CESs are used to expand and differentiate cells. Cell expansion systemsare known in the art. For example, U.S. Pat. No. 5,162,225 and U.S. Pat.No. 6,001,585 generally describe cell expansion systems designed forcell expansion.

The potential of stem cells in a variety of potential treatments andtherapies have achieved particular attention. Stem cells which areexpanded from donor cells can be used to repair or replace damaged ordefective tissues and have broad clinical applications for a wide rangeof diseases. Recent advances in the regenerative medicine fielddemonstrates that stem cells have properties such as proliferation andself-renewal capacity, maintenance of the unspecialized state, and theability to differentiate into specialized cells under particularconditions.

Cell expansion systems can be used to grow stem cells, as well as othertypes of cells. There is a need for cell expansion systems that can beused to grow adherent cells, non-adherent cells, and co-cultures ofvarious cell types. The ability to provide sufficient nutrient supply tothe cells, removing metabolites, as well as furnishing a physiochemicalenvironment conducive to cell growth in a flexible system is an ongoingchallenge. The present disclosure addresses these and other needs.

SUMMARY

In one aspect, the disclosure is directed to a CES including a firstcirculation path having a first fluid flow path with at least opposingends. Opposing ends of the first fluid flow path are fluidly associatedwith opposing ends of a plurality of hollow fibers disposed in a cellgrowth chamber, such that fluid can flow through the first circulationpath in a circuit. A first flow controller is operably linked to thefirst fluid flow path.

The cell expansion system further includes a second circulation path.The second circulation path includes a second fluid flow path with atleast opposing ends. First and second ends of the second fluid flow pathare fluidly associated with the cell growth chamber. A portion of thesecond fluid flow path is in fluid contact with the opposite side of oneor more membranes in the cell growth chamber. A second fluid controlleris operably linked to the second closed circuit path.

The cell expansion system further includes a first fluid supply linefluidly associated with the first circulation path and operably linkedto a third fluid controller. The cell expansion system further includesa first fluid outlet path fluidly associated with the first or secondcirculation path. In various embodiments, the first fluid inlet path orthe first fluid outlet path are operably associated with a third fluidflow controller.

In various embodiments, the CES is configured to allow fluid media inthe first fluid circulation path to flow in a direction opposite to thedirection of fluid media in the second fluid flow path(“counter-current”). Alternatively, the CES is configured to allow fluidmedia in the first fluid circulation path to flow in the same directionas fluid media in the second fluid flow path (“co-current”).

In various additional aspects, the CES can be configured to add media tothe first or second fluid circulation paths without exposing the CES toatmosphere.

In other aspects, the CES further includes an oxygenator. In certainvariations, the oxygenator includes oxygenator inlet and outlet portsdisposed in the oxygenator housing. Oxygenators can be part of the firstcirculation path or second circulation path. Oxygenators thus provideoxygen and/or other gases to the first or second fluid circulationpaths.

In various other aspects, the present disclosure is directed to methodsof expanding cells in the cell expansion system. Generally, cells areadded to the first fluid flow path of the CES. Cells are then incubatedunder appropriate conditions to produce an expanded population of cells.This expanded population can then be harvested.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures illustrate non-limiting exemplary embodiments ofthe CESs disclosed herein, as well as components, uses thereof, and datarelating thereto.

FIG. 1A depicts a flow diagram of one embodiment of a cell expansionsystem.

FIG. 1B depicts a flow diagram of one embodiment of a cell expansionsystem.

FIG. 1C depicts a flow diagram of another embodiment of a cell expansionsystem.

FIG. 1D depicts an embodiment of a CES similar to that of FIG. 1B.

FIG. 1E depicts a rocking device for moving a cell growth chamberrotationally or laterally during operation of the CES.

FIG. 1F depicts an embodiment of a detachable flow circuit.

FIG. 2A depicts a side view of a hollow fiber cell growth chamberembodiment of a cell growth chamber.

FIG. 2B depicts a cut-away side view of the hollow fiber cell growthchamber embodiment of FIG. 2A.

FIG. 3 depicts the mesenchymal stem cells (MSC) doubling time (a) as afunction of the number of colonies in flask prepared samples of MSCs.

FIG. 4 depicts the effect of bone marrow age on number of MSC colonies.

FIG. 5 depicts the effect of bone marrow age on the MSC doubling time(alpha).

FIGS. 6A, 6B, 6C, and 6D depict a flow chart protocol for priming theCES of FIG. 1D.

FIG. 7 depicts a flow chart protocol for draining the CES of FIG. 1D.

FIG. 8 depicts a flow chart protocol for filling drip chamber D1 in theCES of FIG. 1D.

FIGS. 9A and 9B depict a flow chart protocol for exchanging media in thefirst and second fluid circulation paths in the CES of FIG. 1D.

FIG. 10 depicts a flow chart protocol for loading cells into the cellgrowth chamber of the CES of FIG. 1D.

FIG. 11 depicts a flow chart protocol for loading cells into the cellgrowth chamber of the CES of FIG. 1D.

FIG. 12 depicts a flow chart protocol for loading bone marrow cells ontothe CES of FIG. 1D.

FIG. 13 depicts a flow chart protocol for growing cells in the CES ofFIG. 1D.

FIG. 14 depicts a flow chart protocol for harvesting cells from the CESof FIG. 1D.

FIG. 15 depicts a flow chart protocol for harvesting cells from the CESof FIG. 1D.

FIG. 16 depicts a flow chart protocol for rocking the cell growthchamber of the CES of FIG. 1D.

FIG. 17 depicts a flow chart protocol for removing gas from the cellgrowth chamber of the CES of FIG. 1D.

FIGS. 18A, 18B, 18C, 18D, 18E, and 18F depict the expression levels ofcell surface markers tested for using different cell growth protocolsfor cells grown in a hollow fiber bioreactor in the CES of FIG. 1D.

DETAILED DESCRIPTION

The present disclosure is generally directed to cell expansion systemsand methods of using the same.

An exemplary schematic of a cell expansion system (CES) is depicted inFIG. 1A. CES 10 includes first fluid circulation path 12 and secondfluid circulation path 14. First fluid flow path 16 has at leastopposing ends 18 and 20 fluidly associated with a hollow fiber cellgrowth chamber 24 (also referred to herein as a “bioreactor”).Specifically, opposing end 18 is fluidly associated with a first inletport 22 of cell growth chamber 24, and opposing end 20 is fluidlyassociated with first outlet port 28 of cell growth chamber 24. Fluid infirst circulation path 12 flows through the interior of hollow fibers ofhollow fiber membrane 50 disposed in cell growth chamber 24 (cell growthchambers and hollow fiber membranes are described in more detail infra).Further, first fluid flow controller 30 is operably connected to firstfluid flow path 16, and controls the flow of fluid in first circulationpath 12.

Second fluid circulation path 14 includes second fluid flow path 34,cell growth chamber 24, and a second fluid flow controller 32. Thesecond fluid flow path 34 has at least opposing ends 36 and 38. Opposingends 36 and 38 of second fluid flow path 34 are fluidly associated witha second inlet port 40 and a second outlet port 42 respectively of cellgrowth chamber 24. Fluid flowing through cell growth chamber 24 is incontact with the outside of hollow fiber membrane 50 in the cell growthchamber 24. Second fluid circulation path 14 is operably connected tosecond fluid flow controller 32.

First and second fluid circulation paths 12 and 14 are thus separated incell growth chamber 24 by a hollow fiber membrane 50. Fluid in firstfluid circulation path 12 flows through the intracapillary (“IC”) spaceof the hollow fibers in the cell growth chamber. First circulation path12 is thus referred to as the “IC loop.” Fluid in second circulationpath 14 flows through the extracapillary (“EC”) space in the cell growthchamber. Second fluid circulation path 14 is thus referred to as the “ECloop.” Fluid in first fluid circulation path 12 can flow in either aco-current or counter-current direction with respect to flow of fluid insecond fluid circulation path 14.

Fluid inlet path 44 is fluidly associated with first fluid circulationpath 12. Fluid inlet path 44 allows fluid into first fluid circulationpath 12, while fluid outlet path 46 allows fluid to leave CES 10. Thirdfluid flow controller 48 is operably associated with fluid inlet path44. Alternatively, a fourth fluid flow controller (not shown) can beassociated with first fluid outlet path 46.

Fluid flow controllers as used herein can be a pump, valve, clamp, orcombination thereof. Multiple pumps, valves, and clamps can be arrangedin any combination. In various embodiments, the fluid flow controller isor includes a peristaltic pump. In further embodiments, fluidcirculation paths, inlet ports, and outlet ports can be constructed oftubing of any material.

Various components are referred to herein as “operably associated.” Asused herein, “operably associated” refers to components that are linkedtogether in operable fashion, and encompasses embodiments in whichcomponents are linked directly, as well as embodiments in whichadditional components are placed between the two linked components.“Operably associated” components can be “fluidly associated.” “Fluidlyassociated” refers to components that are linked together such thatfluid can be transported between them. “Fluidly associated” encompassesembodiments in which additional components are disposed between the twofluidly associated components, as well as components that are directlyconnected. Fluidly associated components can include components that donot contact fluid, but contact other components to manipulate the system(e.g. a peristaltic pump that pumps fluids through flexible tubing bycompressing the exterior of the tube).

Generally, any kind of fluid, including buffers, protein containingfluid, and cell-containing fluid can flow through the variouscirculations paths, inlet paths, and outlet paths. As used herein,“fluid,” “media,” and “fluid media” are used interchangeably.

Cell Growth Chambers

The cell growth chamber of the cell expansion system generally includesa hollow fiber membrane comprised of a plurality of semi-permeablehollow fibers separating first and second fluid circulation paths.

An exemplary cell growth chamber is depicted in FIG. 2, which depicts acut-away side view of the hollow fiber cell growth chamber 200. Cellgrowth chamber 200 is bounded by cell growth chamber housing 202. Cellgrowth chamber housing 202 further includes four openings, or ports:inlet port 204, outlet port 206, inlet port 208, and outlet port 210.

Fluid in the first circulation path enters cell growth chamber 200through inlet port 204, passes into and through the intracapillary sideof a plurality of hollow fibers (referred to in various embodiments asthe intracapillary (“IC”) side or “IC space” of a hollow fibermembrane), and out of cell growth chamber 200 through outlet port 206.The terms “hollow fiber,” “hollow fiber capillary,” and “capillary” areused interchangeably. A plurality of hollow fibers are collectivelyreferred to as a “membrane.” Fluid in the second circulation path flowsin the cell growth chamber through inlet port 208, comes in contact withthe outside of the hollow fibers (referred to as the “EC side” or “ECspace” of the membrane), and exits cell growth chamber 200 via outletport 210. Cells can be contained within the first circulation path orsecond circulation path, and can be on either the IC side or EC side ofthe membrane.

Although cell growth chamber housing 202 is depicted as cylindrical inshape, it can have any other shape known in the art. Cell growth chamberhousing 202 can be made of any type of biocompatible polymeric material.Various other cell growth chamber housings may differ in shape and size.

Those of skill in the art will recognize that the term cell growthchamber does not imply that all cells being grown or expanded in a CESare grown in the cell growth chamber. In many embodiments, adherentcells can adhere to membranes disposed in the growth chamber, or maygrow within the associated tubing. Non-adherent cells (also referred toas “suspension cells”) can also be grown. Cells can be grown in otherareas within the first or second fluid circulation path.

For example, the ends of hollow fibers 212 can be potted to the sides ofthe cell growth chamber by a connective material (also referred toherein as “potting” or “potting material”). The potting can be anysuitable material for binding the hollow fibers 212, provided that theflow of media and cells into the hollow fibers is not obstructed andthat liquid flowing into the cell growth chamber through the IC inletport flows only into the hollow fibers. Exemplary potting materialsinclude, but are not limited to, polyurethane or other suitable bindingor adhesive components. In various embodiments, the hollow fibers andpotting may be cut through perpendicular to the central axis of thehollow fibers at each end to permit fluid flow into and out of the ICside. End caps 214 and 216 are disposed at the end of the cell growthchamber.

Fluid entering cell growth chamber 200 via inlet port 208 is in contactwith the outside of hollow fibers. This portion of the hollow fiber cellgrowth chamber is referred to as the “extracapillary (EC) space.” Smallmolecules (e.g. water, oxygen, lactate, etc.) can diffuse through thehollow fibers from the interior of the hollow fiber to the EC space, orfrom the EC space to the IC space. Large molecular weight molecules suchas growth factors are typically too large to pass through the hollowfibers, and remain in the IC space of the hollow fibers. The media maybe replaced as needed. Media may also be circulated through anoxygenator to exchange gasses as needed.

In various embodiments, cells can be loaded into the hollow fibers byany of a variety of methods, including by syringe. The cells may also beintroduced into the cell growth chamber from a fluid container, such asa bag, which may be fluidly associated with the cell growth chamber.

Hollow fibers are configured to allow cells to grow in theintracapillary space (i.e. inside the hollow fiber lumen) of the fibers.Hollow fibers are large enough to allow cell adhesion in the lumenwithout substantially impeding the flow of media through the hollowfiber lumen. In various embodiments, the inner diameter of the hollowfiber can be greater than or equal to 10000, 9000, 8000, 7000, 6000,5000, 4000, 3000, 2000, 1000, 900, 800, 700, 650, 600, 550, 500, 450,400, 350, 300, 250, 200, 150, or 100 microns. Likewise, the outerdiameter of the hollow fiber can be less than or equal to 10000, 9000,8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 650, 700,650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 microns.The hollow fiber wall thickness is sufficient to allow diffusion ofsmall molecules.

Any number of hollow fibers can be used in a cell growth chamber,provided the hollow fibers can be fluidly associated with the inlet andoutlet ports of the cell growth chamber. In various embodiments, thecell growth chamber can include a number of hollow fibers greater thanor equal to 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,11000 or 12000. In other embodiments, the cell growth chamber caninclude a number of hollow fibers less than or equal to 12000, 11000,10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, or 2000. In othervarious embodiments, the length of the hollow fibers can be greater thanor equal to 100, 200, 300, 400, 500, 600, 700, 800, or 900 millimeters.In certain embodiments, the cell growth chamber contains approximately9000 hollow fibers that have an average length of 295 mm, an averageinner diameter of 215 microns, and an average outer diameter of 315microns.

Hollow fibers can be constructed of any material capable of forming asize sufficient to form fibers capable of transporting liquid from thecell growth chamber inlet port to the cell growth chamber outlet port.In various embodiments, the hollow fibers can be constructed fromplastic adherent materials capable of binding to certain types of cells,such as adherent stem cells (e.g. MSCs). In various other embodiments,hollow fibers can be treated with compounds such as fibronectin to formadherent surfaces.

In certain embodiments, the hollow fibers may be made of asemi-permeable, biocompatible polymeric material. One such polymericmaterial which can be used is a blend of polyamide, polyarylethersulfoneand polyvinylpyrrolidone (referred to herein as “PA/PAES/PVP”). Thesemi-permeable membrane allows transfer of nutrients, waste anddissolved gases through the membrane between the EC space and IC space.In various embodiments, the molecular transfer characteristics of thehollow fiber membranes are chosen to minimize loss of expensive reagentsnecessary for cell growth such as growth factors, cytokines etc. fromthe hollow fiber, while allowing metabolic waste products to diffusethrough the membrane into the hollow fiber lumen side to be removed.

In certain variations, one outer layer of each PA/PAES/PVP hollow fiberis characterized by a homogenous and open pore structure with a definedsurface roughness. The openings of the pores are in the size range of0.5-3 um, and the number of pores on the outer surface of the fibers arein the range of 10,000 to 150,000 pores per mm². This outer layer has athickness of about 1 to 10 um. The next layer in each hollow fiber is asecond layer having the form of a sponge structure and, in a furtherembodiment, a thickness of about 1 to 15 um. This second layer serves asa support for the outer layer. A third layer next to the second layerhas the form of finger-like structures. This third layer providesmechanical stability and a high void volume which gives the membrane avery low resistance to transporting molecules through the membrane.During use, the finger-like voids are filled with fluid and the fluidgives a lower resistance for diffusion and convection than a matrix witha sponge-filled structure having a lower void volume. This third layerhas a thickness of 20 to 60 um.

In further embodiments, the hollow fiber membrane can include 65-95% byweight of at least one hydrophobic polymer and 5-35% by weight of atleast one hydrophilic polymer. The hydrophobic polymer may be chosenfrom the group consisting of polyamide (PA), polyaramide (PAA),polyarylethersulphone (PAES), polyethersulphone (PES), polysulphone(PSU), polyarylsulphone (PASU), polycarbonate (PC), polyether,polyurethane (PUR), polyetherimide and copolymer mixtures of any of theabove polymers, such as polyethersulphone or a mix ofpolyarylethersulphone and polyamide. In additional embodiments, thehydrophilic polymer may be chosen from the group consisting ofpolyvinylpyrrolidone (PVP), polyethylene glycol (PEG),polyglycolmonoester, water soluble cellulosic derivates, polysorbate andpolyethylene-polypropylene oxide copolymers.

Depending upon the type of cells to be expanded in the cell growthchamber, the polymeric fibers may be treated with a substance, such asfibronectin, to enhance cell growth and/or adherence of the cells to themembrane.

Cell Expansion Systems

Cell growth chambers such as the one depicted in FIG. 2 are operablyassociated with other components of cell expansion systems.

FIG. 1B depicts a more detailed cell expansion system 100. CES 100includes first fluid circulation path 126 (also referred to as the“intracapillary (IC) loop”) and second fluid circulation path 166. Fluidflow paths are constructed of tubing and tubing conduits (Tygothane, St.Globain) and operates in conjunction with valves, pumps and othercomponents.

Outlet port 158 of cell growth chamber 102 is fluidly associated viatubing with inlet port 156, which together with cell growth chamber 102form first fluid circulation path 126. In the embodiment depicted inFIG. 1B, first fluid circulation path 126 is configured for fluid toflow through cell growth chamber 102, sample coil 160, pump 142, andback through cell growth chamber 102. Cells can be flushed out of cellgrowth chamber 102 or redistributed along the hollow fiber membrane.

First fluid circulation path 126 also includes sample coil 160. Samplecoil 160 allows samples of fluid in first fluid circulation path 126 tobe obtained and tested.

CES 100 also includes second fluid circulation path 166 (also referredto as the “extracapillary loop” or “EC loop”). Second fluid circulationpath 166 includes pump 168, temperature meter 170, and oxygenator 104.The second fluid flow path connects to oxygenator inlet port 172 andexits into oxygenator outlet port 174. Oxygenator outlet port 174 isassociated with cell growth chamber 102 by inlet port 162, and departscell growth chamber 102 via cell growth chamber outlet port 164. Secondfluid circulation path 166 is configured for fluid to pass through valve138, into drip chamber 186, and back through pump 168.

Second fluid circulation path 166 provides gas to the cells in cellgrowth chamber 102, and also allows for removal of waste metabolitesproduced by the cells. Gas flows into and out of oxygenator 104 viafilters 150 and 152. Filters 150 and 152 prevent contamination of theoxygenator or associated media. Media flows into oxygenator inlet port172, through fibers contained in oxygenator 104, and leaves throughoutlet port 174. Oxygen enters oxygenator 104 at gas inlet port 176. Theconcentration of gases in the oxygenator can be any concentrationdesired. Gases diffuse across the fibers in the oxygenator.

Fluid media contained in second fluid circulation path 166 is inequilibrium with the gases flowing in through gas inlet port 176. Theamount of oxygen entering the media can be controlled by controlling thegas concentration. The mole percent (also referred to herein as “molarconcentration”) of oxygen in the gas phase before diffusing into themedia is typically greater than or equal to 0%, 5%, 10% or 15%.Alternatively, the mole percent of oxygen in the gas is equal to or lessthan 20%, 15%, 10% or 5%. In certain embodiments, the molarconcentration of oxygen is 5%. Various oxygenators known in the art canbe used as well. Any commercial oxygenator can be used. In certainembodiments, oxygenators have a hollow fiber count of 1820, an internalfiber diameter of 280 μm, an outer fiber diameter of 386 μm and anintracapillary fluid volume of 16 mL.

CES 100 includes first fluid inlet path 124. First fluid inlet path 124includes drip chamber 180 and pump 182. Fluid media and/or cells flowfrom EC media container 106 through valve 107; IC fluid media container108 through valve 109; vent bag 110 through valve 111; or cell input bag112 through clamp 113. Each of IC fluid media container 108, EC mediacontainer 106, vent bag 110, or cell input bag 112 are fluid mediacontainers as discussed herein. IC media generally refers to media thatcirculates in first circulation path 126. EC media generally refers tomedia that circulates in second circulation path 166.

Drip chamber 180 helps prevent pockets of gas (e.g. air bubbles) fromreaching cell growth chamber 102. Ultrasonic sensors can be disposednear entrance port 128 and exit port 130 of drip chamber 180. A sensorat entrance port 128 prevents fluids in drip chamber 180 fromback-flowing into EC media container 106, IC media container 108, ventbag 110, cell input bag 112, or related tubing. A sensor at exit port130 stops pump 182 if gas reaches the bottom of the sensor to preventgas bubbles from reaching cell growth chamber 102.

CES 100 further includes second fluid inlet path 114. When valve 115 isopened, pump 190 can pump fluid from second fluid inlet path 114 intosecond fluid circulation path 166. Connector path 116 connects firstcirculation path and second circulation path. Pump 118 can pump fluidthrough connector path 116 from second fluid inlet path 114 into firstfluid circulation path 126. Alternatively, fluid can be pumped betweenfirst fluid circulation path 126 and second fluid circulation path 166.

Those of skill in the art will recognize that fluid in first fluidcirculation path 126 can flow through cell growth chamber 102 in eitherthe same direction as fluid in second fluid circulation path 166(co-current) or in the opposite direction of second fluid circulationpath 166 (i.e. counter-current).

First fluid circulation path 126 is associated with first fluid inletpath 124 via flush line 132. Flush line includes valve 133, which can beopened and closed in combination with other valves and pumps to flowmedia to or from first fluid inlet path 124.

Likewise, first and second fluid flow paths are connected by fluidconnector path 139. Valve 131 is disposed in fluid connector path 139.By opening valve 131 and using one or more pumps in CES 100, fluid canmove between first fluid circulation path 126 and second fluidcirculation path 166.

Cells can be harvested via cell harvest path 134. Cell harvest path 134is fluidly associated with cell harvest bag 140 and first fluidcirculation path 126 at junction 188. Cell harvest path 134 can beclosed using clamp 117. Cells from cell growth chamber 102 can be pumpedthrough cell harvest path 134 to cell harvest bag 140. Those of skill inthe art will recognize that clamp 117 can be replaced by or combinedwith a valve, pump, or combination thereof in various embodiments.

Various components of the CES can be contained within incubator 199.Incubator 199 maintains cells and media at a constant temperature.

Fluid outlet path 136 is associated with drip chamber 186. Fluid outletpath 136 directs media from drip chamber 186 to waste bag 148.

As used herein, the terms “media bag,” “vent bag” and “cell input bag”are arbitrary, in that their positions can be switched relative to otherbags. For example, vent bag 110 can be exchanged with IC media container108, or with cell bag 112. The input and output controls and parameterscan then be adjusted to accommodate the changes. It will further benoted that the location of the drip chamber, or sensors independent ofthe drip chamber, can be at any location in the CES before inlet port156.

Those of skill in the art will further recognize that the pumps andvalves in the CES of FIG. 1B serve as fluid flow controllers. In variousembodiments, fluid flow controllers can be pumps, valves, orcombinations thereof in any order, provided that the first fluidcirculation path and second fluid circulation path are configured tocirculate fluid and fluid input path(s) are configured to add fluid.

The CES can include additional components. For example, one or more pumploops (not shown) can be added at the location of peristaltic pumps onthe CES. Peristaltic pumps are operably connected to the exterior oftubing, and pumps liquid through the fluid flow path by constricting theexterior of the tubing to push liquid through the tubing. The pump loopsmay be made of polyurethane (PU) (available as Tygothane C-210A),neoprene based material (e.g. Armapure, St. Gobain), or any othersuitable material. Alternatively, a cassette for organizing the tubinglines and which may also contain tubing loops for the peristaltic pumpsmay also be included as part of the disposable. One or more of thecomponents of the CES can be contained in a cassette to aid inorganizing the tubing.

In various embodiments, the CES can include sensors for detecting mediaproperties such as pH, as well as cellular metabolites such as glucose,lactate, and oxygen. The sensors can be operably associated with the CESat any location in the IC or EC loops. Various commercially availablepH, glucose, or lactate sensor can be used.

FIG. 1C depicts another embodiment of a CES. CES 300 includes firstfluid circulation path 302 (also referred to as the “intracapillary (IC)loop”) and second fluid circulation path 304 (also referred to as the“extracapillary loop” or “EC loop”).

First fluid flow path 306 is fluidly associated with cell growth chamber308 to form first fluid circulation path 302. Fluid flows into cellgrowth chamber 308 through inlet port 310, through hollow fibers in cellgrowth chamber 308, and exits via outlet port 307. Pressure gauge 317measures the pressure of media leaving cell growth chamber 308. Mediaflows through valve 313 and pump 311, which can be used to control therate of media flow. Samples of media can be obtained from sample port305 or sample coil 309 during operation. Pressure/temperature gauge 315disposed in first fluid circulation path allows detection of mediapressure and temperature during operation. Media then returns to inletport 310 to complete fluid circulation path 302. Cells expanded in cellgrowth chamber 308 can be flushed out of cell growth chamber 308 orredistributed within hollow fibers for further growth.

Second fluid circulation path 304 includes second fluid flow path 312that is fluidly associated with cell growth chamber 308 in a loop. Fluidin second fluid circulation path 304 enters cell growth chamber 308 viainlet port 314, and leaves cell growth chamber 308 via outlet port 316.Media is in contact with the outside of the hollow fibers in the cellgrowth chamber 308, allowing diffusion of small molecules into and outof the hollow fibers.

Pressure/temperature gauge 319 disposed in the second circulation pathallows the pressure and temperature of media to be measured before themedia enters the EC space of the cell growth chamber 308. Pressure gauge321 allows the pressure of media in the second circulation path to bemeasured after it leaves the cell growth chamber.

After leaving outlet port 316 of cell growth chamber 308, fluid insecond fluid circulation path 304 passes through pump 320 and valve 322to oxygenator 318. Second fluid flow path 312 is fluidly associated withoxygenator 318 via oxygenator inlet port 324 and oxygenator outlet port326. In operation, fluid media flows into oxygenator 318 via oxygenatorinlet port 324, and exits oxygenator 318 via oxygenator outlet port 326.

Oxygenator 318 adds oxygen to media in the CES. In various embodiments,media in second fluid circulation path 304 is in equilibrium with gasentering oxygenator. The oxygenator can be any oxygenator known in theart. Gas flows into oxygenator 318 via filter 328 and out of oxygenator318 through filter 330. Filters 328 and 330 reduce or preventcontamination of oxygenator 318 and associated media.

In the configuration depicted for CES 300, fluid media in firstcirculation path 302 and second circulation path 304 flow through cellgrowth chamber 308 in the same direction (a co-current configuration).Those of skill in the art will recognize that CES 300 can also beconfigured in a counter-current conformation. Those of skill in the artwill recognize that the respective inlet and outlet ports can bedisposed in the cell growth chamber at any location.

Cells and fluid media can be introduced to fluid circulation path 302via first fluid inlet path 332. Fluid container 334 and fluid container336 are fluidly associated with first fluid inlet path 332 via valves338 and 340 respectively. Likewise, cell container 342 is fluidlyassociated with first fluid circulation path 302 via valve 343. Cellsand fluid proceed through heat exchanger 344, pump 346, and into dripchamber 348. Drip chamber 348 is fluidly associated with firstcirculation path 302. Overflow from drip chamber 348 can flow out ofdrip chamber 348 from overflow line 350 via valve 352.

Additional fluid can be added to first or second fluid circulation paths302 and 304 from fluid container 354 and fluid container 356. Fluidcontainer 354 is fluidly associated with valve 358 which is fluidlyassociated with first fluid circulation path 302 via first fluid inletpath 360. First fluid flow path includes valve 364. Alternatively, fluidcontainer 354 is fluidly associated with second fluid inlet path 362.Likewise, fluid container 356 is fluidly associated with valve 366,which is fluidly associated with first fluid circulation path 302 viafirst fluid inlet path 360. Alternatively, fluid container 364 isfluidly associated with second fluid inlet path 362.

Second fluid inlet path 362 is configured to allow fluid to flow throughpump 368 before entering drip chamber 370. Second fluid inlet path 362continues to second fluid circulation path 304. Overflow fluid can flowout via overflow line 372 through valve 374 to waste container 376.

Cells can be harvested via cell harvest path 378. Cells from cell growthchamber 308 can be harvested by pumping media containing the cellsthrough cell harvest path 378 to cell harvest bag 380.

First and second fluid circulation paths 302 and 304 are connected byconnector path 384. When valve 386 is opened, media can flow throughconnector path 384 between first and second circulation paths 302 and304. Likewise, pump 390 can pump media through another connector path388 between first and second fluid circulation paths 302 and 304.

Various components of the CES can be contained within incubator 399.Incubator 399 maintains cells and media at a constant temperature.

As will be recognized by those of skill in the art, any number of fluidcontainers (e.g. media bags) can be fluidly associated with the CES inany combination. It will further be noted that the location of the dripchamber, or sensors independent of the drip chamber, can be at anylocation in the CES before inlet port 310.

The CES can include additional components. For example, one or more pumploops (not shown) can be added at the location of peristaltic pumps onthe CES. The pump loops may be made of polyurethane (PU) (available asTygothane C-210A)). Alternatively, a cassette for organizing the tubinglines and which may also contain tubing loops for the peristaltic pumpsmay also be included as part of the disposable.

Rocking Device

The CES can include a device configured to move or “rock” the cellgrowth chamber relative to other components of the cell expansion systemby attaching it to a rotational and/or lateral rocking device. FIG. 1Eshows one such device, in which a bioreactor 400 is rotationallyconnected to two rotational rocking components, and a lateral rockingcomponent.

A first rotational rocking component 402 rotates the bioreactor aroundcentral axis 410 of the bioreactor. Rotational rocking component 402 isrotationally associated to bioreactor 400. Bioreactor 400 can be rotatedcontinuously in a single direction around central axis 410 in aclockwise or counterclockwise direction. Alternatively, bioreactor 400can rotate in alternating fashion, first clockwise, thencounterclockwise around central axis 410.

The CES can also include a second rotational rocking component thatrotates bioreactor 400 around rotational axis 412. Rotational axis 412passes through the center of point of bioreactor 400 and is normal tocentral axis 410. Bioreactor 400 can be rotated continuously in a singledirection around rotational axis 412 in a clockwise or counterclockwisedirection. Alternatively, bioreactor 400 can be rotated aroundrotational axis 412 in an alternating fashion, first clockwise, thencounterclockwise. In various embodiments, bioreactor 400 can also berotated around rotational axis 412 and positioned in a horizontal orvertical orientation relative to gravity.

Lateral rocking component 404 is laterally associated with bioreactor400. The plane of lateral rocking component 404 moves laterally in the−x and −y directions.

The rotational and/or lateral movement of the rocking device can reducethe settling of cells within the device and reduce the likelihood ofcells becoming trapped within a portion of the bioreactor. The rate ofcells settling in the cell growth chamber is proportional to the densitydifference between the cells and the suspension media according toStoke's Law. In certain embodiments, a 180 degree rotation (fast) with apause (having a total combined time of 30 seconds) repeated as describedabove keeps non-adherent red blood cells suspended (data not shown). Aminimum rotation of about 180 degrees would be preferred; however, onecould use rotation of up to 360 degrees or greater. Different rockingcomponents can be used separately, or can be combined in anycombination. For example, a rocking component that rotates bioreactor400 around central axis 410 can be combined with the rocking componentthat rotates bioreactor 400 around axis 412. Likewise, clockwise andcounterclockwise rotation around different axes can be performedindependently in any combination.

Detachable Flow Circuit

A detachable flow circuit (also referred to herein as a “detachablecirculation module”) is also provided. The detachable flow circuit is aportion of a cell expansion module configured to attach to a fixedportion of the CES. Generally, the fixed portions of the CES includeperistaltic pumps. In various embodiments, the fixed portions of the CEScan include valves and/or clamps.

The detachable flow circuit can include a first fluid flow path havingat least opposing ends. The first end is configured to be fluidlyassociated with a first end of a cell growth chamber, and a second endof the first fluid flow path configured to fluidly associated with anopposing end of the cell growth chamber.

Likewise, the detachable flow circuit can include a second fluid flowpath having at least two opposing ends. Portions of the detachable flowcircuit can be configured to be fluidly associated with an oxygenatorand/or bioreactor. The detachable flow circuit can include a secondfluid flow path that is configured to fluidly associate with theoxygenator and cell growth chamber.

In various embodiments, the detachable flow circuit is detachably anddisposably mounted to a fluid flow controller. The detachable flowcircuit can include detachable fluid conduits (e.g. flexible tubing)that connect portions of the CES. With reference to FIG. 1F, thedetachable flow circuit includes the tubing for first fluid circulationpath 126, but without pump 142. The detachable flow circuit can furtherinclude the tubing for flush line 132, without valve 133. The detachableflow circuit can further include the tubing connecting first circulationpath 126 to flush line 132, and first fluid inlet path 124. In variousother permutations, the detachable flow circuit can include tubing thatconnects the media inlet bags 106 and 108, vent bag 110, and cell inputbag 112 to drip chamber 180. The detachable flow circuit can alsoinclude tubing connecting cell harvest bag 140 to first circulation path126.

Likewise, the detachable flow circuit can include tubing that makes upsecond circulation path 166. For example, the tubing can include tubingconnecting oxygenator 104 to cell growth chamber 102, as well as dripchamber 186. The detachable flow circuit can also include fluid inletpath 114.

In further embodiments, the detachable flow circuit can include a cellgrowth chamber, oxygenator, as well as bags for containing media andcells. In various embodiments, the components can be connected together,or separate. Alternatively, detachable flow circuit can include one ormore portions configured to attach to fluid flow controllers, such asvalves, pumps, and combinations thereof. In variations where peristalticpumps are used, the detachable circuit module can include a peristalticloop configured to fit around a peristaltic portion of the tubing. Invarious embodiments, the peristaltic loop can be configured to befluidly associated with the circulations paths, inlet paths, and outletpaths.

The detachable flow circuit can be combined in a kit with instructionsfor its assembly or attachments to fluid flow controllers, such as pumpsand valves.

Priming the CES

Prior to adding cells, a CES can be “primed” with the media to preparethe CES for operation.

Priming the CES is described in further reference to CES 100 of FIG. 1B.First fluid circulation path 126 is primed before cell growth chamber102 is attached to CES 100. Media is allowed to flow from EC mediacontainer 106 past valve 107, into drip chamber 180 and through firstfluid inlet path 124 to junction 184. Drip chamber 180 is then filledwith media.

First fluid circulation path 126 is then primed with EC media. Media ispumped from drip chamber 180 through flush line 132. Media also flowsthrough first fluid circulation path 126 to junction 184.

Cell growth chamber 102 is then sterile docked into the system byorienting inlet port 156 in a downward direction and outlet port 158 inan upward facing direction. Media is pumped through second fluid inletpath 114 and first fluid inlet path 124. Air is pumped from cell growthchamber 102 to vent bag 110. Specific protocols are described below.

Second fluid circulation path 166 is then primed. Media in second fluidcirculation path 166 flows through oxygenator 104, inlet port 162,outlet port 164, and through oxygenator 104 to complete secondcirculation path 166. Specific protocols are described below.

Variations on priming the CES can be used. For example, the CES can beprimed with the cell expansion system attached to CES 100. Media from ECmedia container 106 or IC media container 108 can be used. Generally,media is added to the system to prevent gas pockets from forming in thesystem.

Media Exchange

Media circulating in either the IC or EC loop can be exchanged withfresh media without removing cells from the CES. IC media can bereplaced with fresh IC media, and EC media can be replaced with fresh ECmedia.

In one embodiment, media can be exchanged by removing used media throughthe hollow fiber membranes in the cell growth chamber (referred toherein as “ultrafiltration”). In various embodiments, ultrafiltration isaccomplished using a cell growth chamber having hollow fibersconstructed from PA/PAES/PVP. With reference to FIG. 1B, fresh media issupplied to drip chamber 180, where it is then allowed to flow tojunction 184. At least a portion of used media leaves first circulationpath 126 by diffusing through the hollow fibers of cell growth chamber102 to enter second circulation path 166 (the “EC loop”). Used mediaenters drip chamber 186, where it is flushed from the system via fluidoutlet path 136 to waste bag 148. Ultrafiltration can be used for bothadherent and non-adherent cells. Generally, large molecules such asproteins are too large to pass through the hollow fiber membranes.Ultrafiltration methods can limit the ability to remove large moleculesfrom the system.

Alternatively, used media can be removed via connector flow path 139.Fresh media is supplied to drip chamber 180. Valve 131 is opened, andused media leaves first circulation path 126 via connector flow path139. The used media is collected in drip chamber 186. Used media is thenflushed from the system via fluid outlet path 136 to waste bag 148. ECmedia can be taken to waste bag 148 through drip chamber 186.

Used EC media can also be exchanged with fresh EC media. With referenceto FIG. 1B, fluid is directed from EC media container 106, through valve115, and directed to the EC loop via pump 190 and pump 168.

Entire volumes of media in the IC loop and EC loop can be readilyexchanged without removing cells from the CES. Small volumes of media orother solution phase compounds can be added to the system as well. Themultiple methods of media exchange further allow media to be exchangedwithout removing cells adhered to the cell growth chamber hollow fibers.

Introducing Cells to the CES

Cells can be added to the CES by a number of methods.

In a first exemplary method, cells can be added to CES 100 byultrafiltration (also referred to as “high flux”), in a similar fashionto media exchange ultrafiltration described above. Cells from cell inputbag 112 are passed through drip chamber 180 with EC and/or IC media at ahigh flow rate to push the cell-containing media into cell growthchamber 102. Subsequently, an excess volume of EC and/or IC media(“chase media”) is loaded into drip chamber 180 and flowed through cellgrowth chamber 102. The chase media can be any media compatible withcells (for example IC media, EC media, or phosphate buffer solution(PBS)). The cells are distributed in hollow fibers of cell growthchamber 102.

Adherent cells (e.g. mesenchymal stem cells, or MSCs) can be selectedbased on adhesion to the hollow fiber lumen. The hollow fiber lumen canbe constructed of an adherent material. Alternatively, the hollow fibercan be treated with fibronectin to cause cell adhesion.

In a second exemplary method, cells can be introduced to the CES by“passively loading” cells onto the media. Cells are introduced from dripchamber 180 into cell growth chamber 102. Media flow is stopped atjunction 188. The volume of the cells and chase media is monitored toensure that cells do not leave first fluid circulation path 126.

Cell Expansion

Cells can be grown (“expanded”) in either the IC loop or the EC loop.Adherent and non-adherent suspension cells can be expanded.

In one embodiment, the lumen of the cell growth chamber fibers can becoated with fibronectin. Divalent cation-free (e.g. calcium andmagnesium-free) PBS is added to the system. After adherent cells areintroduced into cell growth chamber 102, they are incubated for asufficient time to adhere to the hollow fibers. IC and EC media arecirculated to ensure sufficient nutrients are supplied to the cells.

The flow rate of the IC loop and EC loop can be adjusted to a specificvalue. In various embodiments, the flow rate of the IC loop and EC loopscan be, independently, equal to and/or less than 2, 4, 6, 8, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500mL/minute. In various embodiments of the CES of FIG. 1B, the flow ratefor the IC loop is 10-20 mL/minute, and the flow rate of the EC loop is20-30 mL per minute (allowing media to flow through oxygenator 104 andre-establish oxygen levels). Pump 190 can optionally pump additionalmedia into the CES at a low flow rate (e.g. 01. mL per minute) toreplace media that evaporates through the tubes and oxygenator 104. Invarious embodiments, the EC loop removes cellular waste, and the IC loopincludes growth factors in the media.

The CES provides a great deal of flexibility in varying growthconditions and criteria. Cells can be kept in suspension in the IC loopby circulating media continuously. Alternatively, media circulation canbe stopped, causing cells to settle. Fresh media can be added to the ICloop by ultrafiltration to accommodate excess volume without removingcells. EC media circulation allows for exchange of gas, nutrients, wasteproducts, and addition of new media without removing cells.

Expanded cells can include adherent cells, non-adherent cells, or aco-culture of any combination of cells in the art.

Cell Harvest

To harvest adherent cells, the IC and EC media are replaced with mediathat is free of divalent cations (e.g. divalent cation-free PBS).Trypsin is then loaded into first circulation path 126, and allowed toincubate with adherent cells for a period of time (e.g. 9-10 minutes).The trypsin is then flushed from the system. A shearing force is appliedto the cells by increasing the flow rate through cell growth chamber,and adherent cells that are released from the cell growth chamber arepumped via cell harvest path 134 to cell harvest bag 140.

When non-adherent cells are expanded, the cells can be flushed from thecirculating IC loop. Adherent cells remain in cell growth chamber 102,while non-adherent cells are removed.

The CES can be used to perform a variety of cell expansion methods.

In one embodiment, a seeded population of cells can be expanded. Cellsare introduced, or seeded, into the CES. In certain circumstances, thelumen of the hollow fibers can be conditioned to allow cell adhesion.Cells are then added to the cell growth chamber, and adherent cellsadhere to the hollow fibers, while non-adherent cells (e.g. hematopoeticstem cells, or HSCs) do not adhere. The non-adherent cells can beflushed from the system. After incubation for a period of time, theadherent cells can be released and harvested.

Stem cells, progenitor cells, and fully differentiated cells can all beexpanded.

EXAMPLES

The following non-limiting examples illustrate various aspects of CESoperation.

Example 1

50 mL bone marrow was loaded into a hollow fiber growth chamber of theCES depicted in FIG. 1B.

The cell input bag 108 was prepared with 50 mL bone marrow diluted 1:1with IC media and connected drip chamber 180. 300 mL of EC Media wasseparately prepared.

Second circulation path 166 (i.e. EC loop) was conditioned bycirculating EC media in second circulation path 166 while adding a smallamount of EC media to replace fluid that evaporates from the system. Gasconcentration, pH, and temperature of the circulating system weremeasured to insure proper function.

A solution containing bone marrow was then added to the system. A rockeroperably attached to cell growth chamber 102 was turned on to minimizecell loss. Residual bone marrow was washed into the harvest bag toprevent the hollow fibers from clogging and help to rinse residual cellsfrom the drip chamber.

Bone marrow cells were then circulated through the IC loop at 20 mL perminute, and EC media was circulated through the EC loop. A small amountof media was added regularly to both the IC loop and EC loop to preventformation of gas bubbles in the system and tube collapse due toout-gassing.

The media was replaced periodically by media exchange. EC media wasprepared and placed in the EC media container 106. Drip chamber 180 wasdrained and refilled with EC media. EC media was then added to firstfluid circulation path 126 (i.e. the IC loop). Cells were flushed toharvest bag 140 until the EC media was completely removed from dripchamber 180.

IC media was then added to CES 100 by flushing drip chamber 180 threetimes with IC media.

Example 2

MSCs were loaded into the CES.

CES 100 was prepared as described in Example 1. Media was added to bothfirst and second circulation paths 126 and 166 of CES 100. Cell inputbag 112 containing the MSCs suspended in 50 mL of IC media was preparedand attached to drip chamber 180. EC media container 106 was connectedto drip chamber 180 and to second fluid inlet path 114. The cells wereloaded into drip chamber 180. Pump 182 was set to a high flow rate, andcells were allowed to flow into cell growth chamber 102. 50 mL chasemedia of protein-containing IC media was then loaded into drip chamber182 and loaded onto cell growth chamber 102.

EC media was circulated through the second circulation path 166 (the ECloop) at a flow rate of 20 mL/minute. Media in first circulation flowpath 126 (the IC loop) was circulated at a flow rate of 10 mL perminute, and the cell population was allowed to expand.

Example 3

Adherent cells were collected from the CES.

The IC and EC media were replaced with divalent cation-free PBS asdescribed in the media exchange section above. Trypsin was added to cellgrowth chamber 102 and allowed to incubate with adherent cells for 9-10minutes. The trypsin was flushed very quickly from the system, and ashearing force was applied to the hollow fibers in cell growth chamber126 by increasing the flow rate. The adherent cells released from thehollow fibers were flushed from cell growth chamber 126 by stopping thefirst circulation path and pumping the cell-containing media to harvestbag 140.

To harvest non-adherent cells, no trypsin was added, and cells wereflushed from the first circulation path 126 to cell harvest bag 140.Adherent cells remained adhered to hollow fibers in the cell growthchamber, while non-adherent cells were collected.

Example 4

Non-adherent cells are loaded into the CES.

With reference to FIG. 1B, the cells are loaded in media from cell inputbag 112 into drip chamber 180. The cells are then pumped slowly by pump182 into cell growth chamber 102. The flow rate is adjusted to allow thecells to settle in the hollow fibers of the cell growth chamber 102. Theflow rate is stepped down to steadily decreasing pressures as cells areloaded until all cells are pumped from drip chamber 180 into cell growthchamber 102.

Alternatively, non-adherent cells are loaded into the CES byultrafiltration. The cells moved from cell input bag 112 into dripchamber 180. Pump 182 pumps cells into and through cell growth chamber102 into first circulation path 126.

Subsequently, the cells are concentrated in the cell growth chamber bymedia exchange. IC media is added to first circulation path 126 fromdrip chamber 180. Pump 182 pumps media into cell growth chamber 102through inlet port 156. Simultaneously, pump 142 pumps media into cellgrowth chamber 102 through outlet port 158. Excess media diffusesthrough the hollow fibers into second circulation path 166. Non-adherentcells are thus concentrated in the hollow fibers of cell growth chamber102 by flowing media into the cell growth chamber 102 from bothdirections.

Flow rates can be tested using synthetic beads capable of entering thehollow fibers of the cell growth chamber. The beads can have differentsizes and densities corresponding to different cell types. Thecirculation rate was reduced slowly, and the distribution of beads incell growth chamber 102 was measured to determine if the flow ratechanged bead distribution.

In various embodiments, adherent and non-adherent cells can be grownsimultaneously. In one alternative, adherent cells are loaded andallowed to adhere to cell growth chamber 102. Suspension cells are thenadded to first circulation path 126. In a second alternative, suspensioncells are loaded into cell growth chamber 102 first, and allowed to coatthe surface of the hollow fibers in cell growth chamber 126, therebypromoting adherent cell growth and/or attachment. In a thirdalternative, the suspension cells are added to the CES first, andallowed to grow for a specific period of time. Suspension cells are thenremoved, and adherent cells are added to the system and allowed toadhere to the hollow fibers of the CES. Fresh suspension cells are thenadded, and grown simultaneously with the adherent cells.

In other alternatives, suspension cells and adherent cells are grown ondifferent sides of the cell growth chamber. For example, adherent cellscan be grown on the IC side, and suspension cells on the EC side, orvice versa. Adherent and suspension cells (e.g. MSCs and HSCs) can thusbe maintained separately, but kept in fluid communication with eachother across the membrane. Adherent cells and suspension cells can alsobe grown together on both the IC and EC sides of the cell growthchamber.

Example 5

Non-adherent cells were expanded in a CES.

A series of protocols were developed for various functions of a hollowfiber CES embodiment depicted in FIG. 1D (the CES embodiment depicted inFIG. 1D is substantially similar to the CES embodiment depicted in FIG.1B). FIG. 1D discloses an embodiment having specific pumps (P1-P5),valves (V1 a, V1 b, V2 a, V4, V6, V7, V8 and V9), clamps (C1 and C2),sample ports (S1-S3), drip chambers (D1 and D2), temperature gaugesT1-T3, and pressure gauges PR1 and PR2. Various protocols were developedfor priming, draining and filling drip chambers, exchanging fluid mediabetween first and second fluid circulations paths, loading, expanding,harvesting cells, and removing air from the system.

FIGS. 6A-17 depict flow diagrams that show exemplary processes of usingthe CES. Each block diagram depicts pressure in units of mL per minute,opening and closing of valves, pressure gauges, and connecting ofvarious components.

FIGS. 6A, 6B, 6C, and 6D depict a flow diagram protocol for priming theCES of FIG. 1D. The CES was first primed with EC media. EC media waspumped from the EC media bags into the EC loop by pumps P3 and P1. Dripchamber D1 was filled with EC media. Pumps P4 and P5 pumped EC mediathrough the IC loop, and excess EC media flowed into drip chamber D2.The bioreactor was attached to the system, oriented vertically withrespect to gravity, and pumps P3 and P5 filled the IC loop (includingthe bioreactor) and the EC loop with EC media. The waste bag was hungabove the CES, and pumps P2 and P3 pumped media into the EC loop. PumpsP1, P2, P3 and P5 (at a high flow rate) then pumped media through thesystem, and excess media was allowed to flow through valve V7 to dripchamber D2.

FIG. 7 depicts a flow diagram protocol for adding media from dripchamber D1 into the CES of FIG. 1D. Drip chamber D1 was filled withmedia chosen from any source, including IC media, EC media, and cells.One of three individual protocols can then be performed. In the firstprotocol, new media from drip chamber D1 was pumped through valve V6 inthe IC loop, with excess media leaving the IC loop via valve V7. In thesecond protocol, media was pumped through the bioreactor, and exited thesystem via valve V7 and into drip chamber D2. In the third protocol, newmedia replaced used media via ultrafiltration, by pumping media at ahigh flow rate through the hollow fiber membrane in the bioreactor, anddraining media from the system through valve V8 and into drip chamberD2. Media in drip chamber D2 then was allowed to flow to the waste bag.

FIG. 8 depicts a protocol for filling drip chamber D1. Either IC media,EC media, or cells contained in media were added from the IC media bag,EC media bag, or the cell input bag respectively. The valve or clampseparating drip chamber D1 from the bag containing the chosen medium orcell was opened. The medium or cells were then allowed to flow into dripchamber D1. When drip chamber D1 was filled, the valve to the IC media,EC media, or cell input bag was closed.

FIGS. 9A and 9B depict a flow diagram protocol for media exchangebetween EC and IC media in the CES of FIG. 1D. Valve V8, and optionallyvalves V7 and V9, were opened. IC or EC media was then pumped from dripchamber D1 by pumps P2, P3, P4, and P5. The pump rate ratios of P2/P3and P4/P5 were selected. Specific IC volumes were pumped into the ICloop by pump P5. Alternatively, specific EC volumes were pumped into theEC loop by pump P3. The new volume of media was flushed from dripchamber D1 by pump P5 through valve V8 to junction A in the IC loop.Alternatively, a specific volume of media (in this case 13.5 mL) wasadded to the system. The pumps were then turned on, and the media wascirculated.

FIG. 10 depicts a flow diagram protocol for loading cells on thebioreactor by the ultrafiltration method in the CES of FIG. 1D. Clamp C1was opened, and cells were placed in drip chamber D1. Pump P5 pumped thecells and media from drip chamber D1 into the IC loop. Excess mediaflowed through the hollow fiber membranes, and departed the system viadrip chamber D2. IC or EC chase media were selected, and valves V2 a (toIC media bag) or V1 b and V1 a (to EC media bag) were opened. The pumpsare stopped once the chase media was loaded onto the system.

FIG. 11 depicts a flow diagram protocol for loading cells on thebioreactor by a “passive loading” method. Pump P5 was turned on, IC orEC chase media were selected, and valves V2 a (to IC media bag) or V1 band V1 a (to EC media bag) were opened. Clamp C1 to cell input bag wasopened. Pump P5 pumped at a slow rate to pump cells and associated mediafrom drip chamber D1 to the bioreactor. Excess media flowed out of theIC loop via valve V7 to drip chamber D2. Subsequently, chase IC or ECmedia was loaded onto the system, and excess media flowed out of the ICloop through valve V5.

FIG. 12 depicts a flow diagram protocol for a bone marrow washout in theCES of FIG. 1D. The pressure of pump P5 was set at a high flow rate, andpumps P2 and P3 were set at low flow rates relative to P5. Media fromthe cell input bag, IC media bag, or EC media bag was then added to dripchamber D1. Cell harvest bag was opened by releasing clamp C2. Pumps P2,P3, and P5 pumped cells and associated media into the cell harvest bag.The cell harvest bag was sealed and removed, and V8 and V9 werereopened.

FIG. 13 depicts a flow diagram protocol for growing adherent cells inthe CES of FIG. 1D. Valve V8, and optionally valves V7 and V9, wereopened. IC or EC media was then pumped from drip chamber D1 by pumps P2,P3, P4, and P5. Volumes of IC and EC media were then selected. The ICand EC media were pumped by either P3 or P5 onto the system. Valves V8and V9 were then opened.

FIG. 14 depicts a flow diagram protocol for harvesting adherent ornon-adherent cells from the first circulation path (i.e. the IC loop,including the bioreactor and pump P4) of the CES of FIG. 1D. Pumps P2,P3, P4 and P5 pumped media at selected flow rates. Valve V6 was openedand clamp C2 (to the cell harvest bag) was opened. A harvest volume wasselected, and pumps were started for the period of time necessary topump cells to the harvest bag. Clamp C2 was closed, and the cell harvestbag was removed.

FIG. 15 depicts a flow diagram protocol for harvesting cells adhered tothe bioreactor in the CES of FIG. 1D. Pumps P2, P3 and P5 were selectedat relative flow rates. Valve V8 was opened and clamp C2 (to the cellharvest bag) was opened. A harvest volume was selected, and pumps werestarted for the period of time necessary to pump the harvest volume.Clamp C2 was closed, and the cell harvest bag was removed.

FIG. 16 depicts a flow diagram protocol for controlling a rockerattaching the bioreactor in the CES of FIG. 1D. The rocker rotated thebioreactor for a pre-selected period of time, and/or can be set for apre-selected “dwell time.”

FIG. 17 depicts a flow diagram protocol for removing air from the CES ofFIG. 1D. Pump P5 was started in reverse, and media flows through thehollow fibers by ultrafiltration or through the bioreactor via valve V7.Media was collected in drip chamber D1.

Example 6

The initial adhesion and growth rate of mesenchymal stem cells (MSCs) ina hollow fiber cell growth chamber was studied using two sources ofMSCs.

The recovery of MSCs from fresh bone marrow collected after 2 days ofgrowth was measured to estimate the number of cells remaining in thecell growth chamber after trypsinization and harvest. Cells remaining inthe cell growth chamber were lysed and the LDH mass was measured. LDHmass was compared to a standard LDH per MSC lysed) to estimate themaximum number of MSCs left in the cell growth chamber. This method gavean approximation of the number of MSCs remaining in the cell growthchamber.

Cells were counted using an automated cell counter. Cell viability wasdetermined by dye (trypan blue or erythrosine B) exclusion assay or onan automated counter with viability measurement capabilities. Thephenotype of the cells was detected in post-CES harvest, single colorflow cytometric determination using CD34, CD45, CD90, CD105, CD73, andHLA-DR with respective isotype control on a FACS flow cytometer. Cellmorphology of cells was determined post-harvest. 5,000 cells/cm² wereseeded in 24-well plates (n=3). The morphology of the cells was observedon day 1 after adhesion and 4 days later.

A four-site randomized study testing three cell growth chambers and twoMSC sources was conducted. The three cell growth chambers weretested: 1) a 1.7 m² PA/PAES/PVP membrane, 2) a 1 m² 0.5% Desmopanmembrane, and 3) a 1 m² 2% Desmopan membrane. The two MSC sources werea) direct seeding of bone marrow (to be tested on all three cell growthchambers) and b) pre-selected plastic adherent MSCs from bone marrow (tobe tested only on the first two cell growth chambers).

The study design observed the early attachment and growth of the MSCs,and particularly the initial seeding, attachment, and growth in the cellgrowth chamber. The measured elements included:

1. Initial MSC binding efficiency.

2. MSC doubling time (α).

3. Assuming constant α and initial MSC binding, the projected number ofdays to a therapeutic dose.

4. Quality of the MSCs, specifically purity, phenotype, and MSCdifferentiation.

The hollow fiber membrane and MSC source options are shown in Table 1.The 2% Desmopan membrane was tested only with direct seeded bone marrow(i.e. bone marrow that had not been previously selected for MSCs). Theother membranes were tested with both direct seeded bone marrow and withpre-selected adherent (T-75) MSCs from bone marrow.

TABLE 1 Site Experiment Test 2% Desmopan (direct seed) Site 4 0.5%Desmopan (direct seed and T75) Fnx2 PA/PAES/PVP(direct seed and T75)Test 2% Desmopan (direct seed) Site 3 0.5% Desmopan (direct seed andT75) Fnx2 PA/PAES/PVP (direct seed and T75) Test 2% Desmopan (directseed) Site 2 0.5% Desmopan (direct seed and T75) Fnx2 PA/PAES/PVP(directseed and T75) Test 2% Desmopan (direct seed) Site 1 0.5% Desmopan(direct seed and T75) Fnx2 PA/PAES/PVP(direct seed and T75)

The CES depicted in FIG. 1B was used for the study. The incubatorenclosing the CES was set at 38° C. Table 2 depicts the protocol thatwas followed for each cell growth chamber. The protocol was identical ateach test site.

TABLE 2 Protocol Summary 2% Desmopan 0.5% Desmopan Direct Direct T75Fnx2 PA/PAES/PVP Day Seeding Seeding seeding Direct Seeding T75 Seeding−1 Fibronectin coated PA/PAES/PVP - 2xFn 0 Load ~30 mL Load Plate twoLoad ~50 mL bone Plate two T75 flasks. bone ~30 mL T75 flasks. marrow.marrow. bone marrow. 2 Wash and Wash Wash and Wash and feed CES Wash andfeed T-75 feed CES and feed T75 feed CES 5 Wash and Wash Wash and Washand feed CES Wash and feed T-75 feed CES and feed T75 feed CES 8 Washand Wash Wash and Wash and feed CES Wash and feed T-75 feed CES and feedT75 feed CES 11 Wash and Wash Wash and Wash and feed CES Wash and feedT-75 feed CES and feed T75 feed CES 13 Harvest Harvest Harvest and countFibronectin coat and count and PA/PAES/PVP - 2xFn count 14 HarvestHarvest T75 and load T75 and into CES load into CES 17 Wash and Wash andfeed CES feed CES 19 Wash and Wash and feed CES feed CES 21 HarvestHarvest and count and count

Table 3 shows data for MSCs prepared in a T-25 flask. The Test Site 3bone marrow products were collected at Test Site 3 and used in the CESon Day 0. Test Site 4 bone marrow products were collected by Test Site 3and shipped overnight to Test Site 4 where they were used in the CES onDay 1. Both Test Site 1 and Test Site 2 used commercially purchased bonemarrow products shipped overnight and used in the CES on Day 1.

On the day of use in the CES, two 78 μL samples of bone marrow productwas plated respectively in two T-25 flasks. The number of observablecolonies, total MSCs, and MSC viability were measured seven days afterplating.

To understand the effect of overnight storage, the data was measured onboth pre-expansion days (specifically by Test Site 3 on bone marrowproducts sent to Test Site 4) and on post-run days (specifically postruns which did not use the entire 50 mL bone marrow products, i.e. the 1m² Desmopan cell growth chambers).

TABLE 3 T-25 Cell Count Data T25 Flask Data Days post collection 0 1 2Sample # MSC Viability # MSC Viability # MSC Viability # Colonies Count(%) Colonies Count (%) Colonies Count (%) 2% Test A 19 27600 80.2 1948000 89.6 Desmopan Site 1 B 14 28500 82.8 17 55000 91.5 Test A 13 2400095.0 Site 2 B 11 13000 98.0 Test A 150 140000 100.0 106 115000 100.0 1088310 100.0 Site 3 B 150 230000 100.0 140 140000 100.0 124 9450 100.0Test A 111 74 17420 95.0 63 21780 95.0 Site 4 B 86 73 17790 95.0 9133540 95.0 0.5% Test A 29 38000 93.4 19 15300 89.3 Desmopan Site 1 B 3439300 96.1 16 17600 85.9 Test A 56 222000 88.3 33 19500 56.0 Site 2 B 50149000 80.9 25 28000 73.4 Test A 83 4950 100.0 46 1870 100.0 64 2400100.0 Site 3 B 70 4500 100.0 47 1870 100.0 67 2550 100.0 Test A 16 310 160 95.0 Site 4 B 7 140 2 34 95.0 PA/PAES/ Test A 56 55000 94.5 PVP Site1 B 48 36600 88.7 Test A 25 360000 87.0 Site 2 B 29 280000 88.0 Test A32 1350 100.0 Site 3 B 43 1680 100.0 Test A 185 127 16710 95.0 Site 4 B184 160 166 30 95.0

The protocol defined that for each cell growth chamber to be seeded withadherent pre-selected MSCs, two T-75 flasks would be plated with 3×78 μL(234 μL) of bone marrow. Fourteen days after plating, both T-75 flaskswere harvested using standard trypsin techniques. One harvest was usedto seed the cell growth chamber and the second harvest was counted. (Toreduce the possibility of contamination during open events, the sampleswere not mixed and then divided.)

These data are shown in Table 4.

TABLE 4 Directly Seeded Cell Cell growth chamber with pre- T75 Flasksgrowth chamber selected MSCs (flask grown) MSC MSC LDH Results MSC LDHResults Count on Viability Count Viability (Calc # Count Viability (Calc# day 14 (%) Day 13 (%) of Cells) Day 7 (%) of Cells) 2% Test Site 11.08E+07 93.0 2.97E+05 Desmopan Test Site 2 4.37E+06 50.8 <DL Test Site3 6.30E+06 73.0 2.35E+05 Test Site 4 3.41E+06 97.2 2.31E+05 0.5% TestSite 1 2.86E+06 95.3 1.01E+07 87.9 1.67E+06 1.04E+07 91.2 2.93E+05Desmopan Test Site 2 4.22E+06 90.0 1.38E+07 35.9 4.75E+06 95.0 Test Site3 1.43E+06 100.0 6.23E+06 91.1 3.75E+05 4.20E+06 92.1 <DL Test Site 47.05E+05 95.1 1.62E+06 9.47E+05 1.05E+06 96.9 3.47E+04 PA/PAES/ TestSite 1 3.93E+06 97.5 3.45E+07 92.3 3.10E+06 6.50E+07 88.9 0.00E+00 PVPTest Site 2 1.50E+06 84.0 8.00E+06 66.5 9.25E+07 93.8 Test Site 38.50E+05 100.0 1.58E+07 90.5 2.12E+06 1.87E+07 92.0 <DL Test Site 44.87E+06 94.9 6.32E+07 98.2 1.33E+06 5.83E+07 98.2 1.16E+06 Where DL isthe assay detection limit. DL for Test Site 3 = 5.4E+04

Table 4 depicts MSC count data for T-75 flasks, Directly Seeded Cellgrowth chambers, and Cell growth chambers with Pre-Selected MSCs. Table4 also shows the count data for the direct seeded cell growth chamberharvests and the count data for the cell growth chamber harvests withpre-selected MSCs. All directly seeded cell growth chambers were able toproduce viable MSCs, though Test Site 2 produced lower quantities ofMSCs. A lower quantity of cells was measured at Test sites 1 and 4 usingthe 0.5% desmopan as compared to the T75 flask preparation method.However, all cells had high viability. Test site 4 produced a comparablequantity of highly viable MSCs as compared to the T75 flasks.

Various stem cell markers were used to detect stem cells in each of the2% Desmopan, 0.5% Desmopan, and PA/PAES/PVP hollow fiber membranes.Tables 5, 6, and 7 show phenotype data for the harvests.

TABLE 5 0.5% Desmopan % Positive Site CD34 CD45 CD73 CD90 CD105 HLA-DRDirectly Seeded Bioreactor Test Site 1 0.8 25.0 70.2 72.0 91.8 28.5 TestSite 2 29.5 61.0 18.3 Test Site 3 0.0 14.3 75.1 66.3 79.6 6.4 Test Site4 0.4 0.2 29.7 41.9 72.1 21.9 Average: 7.7 25.1 48.3 60.1 81.2 18.9Standard Dev: 14.6 26.0 28.5 16.0 9.9 11.3 Bioreactor with Pre-SelectedMSCs Test Site 1 0.5 0.3 96.3 97.7 95.7 0.5 Test Site 2 1.3 3.6 97.687.9 91.7 1.2 Test Site 3 0.0 0.0 93.6 90.3 80.5 0.0 Test Site 4 0.2 1.697.5 94.6 82.6 1.4 Average: 0.5 1.3 95.8 91.9 89.3 0.6 Standard Dev: 0.61.6 1.9 4.4 7.3 0.7

TABLE 6 Phenotype data for 0.5% Desmopan harvests. 2% Desmopan DirectlySeeded Bioreactor % Positive Site CD34 CD45 CD73 CD90 CD105 HLA-DR TestSite 1 0.5 87.6 2.3 0.3 78.4 10.8 Test Site 2 7.8 33.9 9.0 15.0 7.5 17.8Test Site 3 0.0 13.8 81.7 76.5 85.4 3.9 Test Site 4 0.1 17.8 82.2 71.094.6 7.4 Average: 2.1 38.3 43.8 40.7 66.5 10.0 Standard Dev: 3.8 34.044.1 38.7 39.9 5.9

TABLE 7 Phenotype data for PA/PAES/PVP Membrane harvests. Fnx2PA/PAES/PVP % Positive Site CD34 CD45 CD73 CD90 CD105 HLA-DR DirectlySeeded Bioreactor Test Site 1 0.3 12.0 90.8 85.8 91.3 6.4 Test Site 21.0 7.8 93.6 98.8 97.0 17.9 Test Site 3 0.0 43.9 53.1 50.2 76.5 25.8Test Site 4 0.1 14.0 84.3 79.7 95.1 56.6 Average: 0.3 19.4 80.4 78.689.9 26.7 Standard Dev: 0.5 16.5 18.6 20.6 9.3 21.5 Bioreactor withPre-Selected MSCs Test Site 1 0.1 0.2 98.5 99.3 96.2 0.5 Test Site 2 2.14.1 83.8 92.0 99.7 0.2 Test Site 3 0.0 0.0 93.4 92.9 95.4 0.0 Test Site4 0.1 0.1 99.6 99.3 97.5 0.2 Average: 0.6 1.5 91.9 94.7 97.1 0.2Standard Dev: 1.0 2.0 7.2 4.0 1.9 0.2

Table 8 shows the calculated MSC doubling times (α, alpha) for the T-25flask data. Also included in this table is the T-25 flask data using 78μL of bone marrow collected during a direct plating stem cell study.These data are plotted in FIG. 3.

For bone marrow products which yield around 50 colony forming units(CFUs) or less, the data is clustered in two groups. With the exceptionthat a majority of those data with the higher alpha are from Test Site3, whereas those data from the lower group are predominately from sitesother than Test Site 3, no common characteristic is obvious.

The T-25 flask data showing the effect of bone marrow age on both CFUsand on the MSC doubling time is graphed in FIG. 4 and FIG. 5,respectively. These data correspond to plating 78 μL of bone marrow andcounting the CFUs and total MSCs seven days later.

FIGS. 18A, 18B, 18C, 18D, 18E, and 18F depict the expression levels ofcell surface markers tested for using different cell growth protocolsfor cells grown in a hollow fiber bioreactor in the CES of FIG. 1D.“Direct” refers to cells loaded directly on the bioreactor. “Pre-sel”refers to cells grown in a flask and pre-selected for MSCcharacteristics that are then grown using a hollow fiber bioreactor.0.5% refers to cells grown on hollow fiber bioreactors loaded with 0.5%Desmopan. 2% refers to cells grown in bioreactors loaded with 2%Desmopan. PA/PAES/PVP refers to cells grown using a PA/PAES/PVP hollowfiber bioreactor.

TABLE 8 Calculation of Alpha for T-25 flask. D E F G H I T25 Flask DataT25 Flask day 7 Data 0.078 ml from 50 ml Bone Marrow Aspirate # Δt αAverage # Average Colonies MSC Count (days) (hrs) colonies α (hrs) 2%Test Site A 19 2.76E+04 7 16.0 16.5 15.6 Desmopan 1 B 14 2.85E+04 7 15.3Test Site A 13 2.40E+04 7 15.5 12.0 16.0 2 B 11 1.30E+04 7 16.5 TestSite A 150 1.40E+05 7 17.0 150.0 16.5 3 B 150 2.30E+05 7 15.9 Test SiteA 74 1.74E+04 7 21.3 73.5 21.3 4 B 73 1.78E+04 7 21.2 0.5% Test Site A29 3.80E+04 7 16.2 31.5 16.4 Desmopan 1 B 34 3.93E+04 7 16.5 Test Site A56 2.22E+05 7 14.1 53.0 14.3 2 B 50 1.49E+05 7 14.6 Test Site A 834.95E+03 7 28.5 76.5 28.2 3 B 70 4.50E+03 7 28.0 Test Site A 1 6.00E+017 28.4 1.5 34.8 4 B 2 3.40E+01 7 41.1 PA/PAES/ Test Site A 56 5.50E+04 716.9 52.0 17.2 PVP 1 B 48 3.66E+04 7 17.5 Test Site A 25 3.60E+05 7 12.227.0 12.4 2 B 29 2.80E+05 7 12.7 Test Site A 32 1.35E+03 7 31.1 37.531.4 3 B 43 1.68E+03 7 31.8 Test Site A 127 1.67E+04 7 23.9 143.5 24.5 4B 160 1.66E+04 7 25.1 Test Site A 19 5.03E+02 7 35.5 14.7 35.3 3 DirectB 19 4.46E+02 7 36.9 Seeding C 6 1.97E+02 7 33.4 Study A 46 1.24E+03 735.3 45.7 33.6 B 39 1.30E+03 7 33.2 C 52 1.91E+03 7 32.3 A 33 9.46E+02 734.7 39.0 34.2 B 41 1.27E+03 7 33.9 C 43 1.34E+03 7 33.9 A 24 8.40E+02 732.8 27.0 34.1 B 28 9.16E+02 7 33.4 C 29 7.30E+02 7 36.1

I claim:
 1. A detachable flow circuit configured to attach to a fixedportion of a cell expansion system comprising a plurality ofcontrollers, the detachable flow circuit comprising: a first fluidcirculation path comprising a first fluid flow path having at leastopposing ends, a first end of the first fluid flow path configured tofluidly associate with a first inlet port of a cell growth chamber and asecond opposing end of the first fluid flow path configured to fluidlyassociate with a first outlet port of the cell growth chamber wherein aportion of the first fluid circulation path is configured to bedisposably mounted to a first fluid controller of the fixed portion ofthe cell expansion system; a second fluid circulation path comprising asecond fluid flow path having at least opposing ends, one end of thesecond fluid flow path configured to fluidly associate with a secondinlet of the cell growth chamber and a second opposing end of the secondfluid flow path configured to fluidly associate with a second outlet ofthe cell growth chamber, wherein a portion of the second fluidcirculation path is configured to be disposably mounted to a secondfluid controller of the fixed portion of the cell expansion system; afirst fluid inlet path fluidly associated with the first fluidcirculation path; a first fluid outlet path fluidly associated with atleast one of the first or second fluid circulation paths; a first fluidconnector path having at least opposing ends, a first opposing end ofthe first fluid connector path fluidly associated with the first fluidcirculation path and a second opposing end of the first fluid connectorpath fluidly associated with the second fluid circulation path, whereina portion of the first fluid connector path is configured to bedisposably mounted to a third fluid controller of the fixed portion ofthe cell expansion system; and a second fluid connector path havingopposing ends, one end of the second fluid connector path fluidlyassociated with the first fluid flow path and a second end of the secondfluid connector path fluidly associated with the second fluid flow path,wherein a portion of the second fluid connector path is configured to bedisposably mounted to a fourth fluid controller of the fixed portion ofthe cell expansion system.
 2. The detachable flow circuit of claim 1,wherein the first fluid controller, the second fluid controller, thethird fluid controller, and the fourth fluid controller each areindependently selected from the group consisting of: a pump, a valve,clamps, and combinations thereof.
 3. The detachable flow circuit ofclaim 2, wherein the cell growth chamber is configured to allow media inthe first fluid flow path to flow in the opposite direction than thesecond fluid flow path in the cell growth chamber.
 4. The detachableflow circuit of claim 1, further comprising: an oxygenator; and tubingconnecting the oxygenator to the cell growth chamber.
 5. The detachableflow circuit of claim 1, further comprising one or more bags, whereinthe one or more bags store one or more from the group consisting of:media and cells.
 6. The detachable flow circuit of claim 1, wherein thecell growth chamber is configured to be operably attached to a rocker onthe fixed portion of the cell expansion system, and wherein the cellgrowth chamber is rotatable by the rocker around a central axis of saidcell growth chamber.
 7. The detachable flow circuit of claim 1, whereinthe cell growth chamber is configured to be operably attached to arocker on the fixed portion of the cell expansion system, and whereinthe cell growth chamber is rotatable by the rocker around an axis normalto a central axis of said cell growth chamber.
 8. The detachable flowcircuit of claim 1, wherein fluid in the first fluid circulation pathflows through an intracapillary space of one or more hollow fibers inthe cell growth chamber.
 9. The detachable flow circuit of claim 1,wherein fluid in the second fluid circulation path flows through anextracapillary space of one or more hollow fibers in the cell growthchamber.